The present invention relates to an enzyme biosensor intended for the direct measurement of biochemical parameters of the skin, as well as to the corresponding method of measurement and the method for preparation of the necessary enzymatic membrane.
A large number of techniques are known for the in vivo measurement, in a non-invasive manner, of physical parameters of the skin. While considerable data is available on these physical parameters, few, however, are available about the biological signals which are directly accessible on the stratum corneum, and methods for measurement providing the direct acquisition of such signals are practically non-existent at the present time.
These biochemical factors are, however, interesting from a diagnostic point of view. Thus, for example, the energetic metabolism of the skin comprises a preponderant anaerobic cycle with formation of tactic acid from pyruvic acid; this lactic acid is found in the suprabasal layers of the epidermis, as well as in the stratum corneum. One of the roles described for the exogenous lactate is that of natural hydrating agent for the skin due to its hygroscopic properties (Van Scott et al, J. Am. Acad. Dermatol. 1984, 11. 869; and Takahashi et al., J. Soc. Cosmet. Chem. 1985, 36, 177). In addition, it is known that an increase in the amount of lactate is a sign of cell hyperproliferation in the deep layers.
The interest of a device providing, under the best conditions, a measurement of the lactate on the skin would, in particular, therefore, be to be able to determine the importance of this metabolite on the hydration of the skin and its possible diagnostic or curative role. The best conditions, of which it is question, are those of direct measurement, without a sampling step and without placing a reagent on the skin. In addition, it could be interesting to be able to carry out repeated measurements on a same site,for example, at one minute intervals, so as to observe phenomena of exhaustion or of facilitated diffusion of the cutaneous lactate.
In reality, it would be generally useful to have means for direct measurement on the cutaneous coating of all dermatologically or cosmetically interesting molecules,such as, for example, urea, cholesterol or amino acids. The studies to be carried out can further, also in a general manner, relate to the remanence, the absorption and the release of one of the compounds in question.
However, taking lactate as an example, the normal methods for determination of this substance, outside of the fact of requiring a sampling step, are sensitive but relatively slow (Barker et al., J. Biol. Chem. 1941, 138, 535). The same disadvantages can be attributed to the enzymatic method using dehydrogenase lactate, hereinafter called LDH (E.C. 1.1.1.27), which is based on the detection at 340 nm of the coenzyme, nicotinamide-adenine-reduced dinucleotide; moreover, interference related to the presence of LDH effecters in the sample can be detrimental to the sensitivity of the measurement (Gutman et al., in H.U. Berymeyer (Ed.), Methods of Enzymatic Analysis, Verlag Chemie, Weinham, 2nd Edition 1974, 264).
In addition, the electrochemical sensors, combined with a film, in which an enzyme is immobilized, have already been widely used to determine compounds found in biological media. Thus, there exists at the present time a large number of L-lactate specific enzymatic electrodes which are used in the field of agro-food industries, as well as in that of medical analyses. Enzyme electrodes using one of four enzymatic systems which can be envisaged for such applications have been described:
LDH [Durliatt et al. Anal. Chem. 1980,52, 2109];
cytochrome b2 (E.C. 1.1.2.3) [Shinbo et al. Anal. Chem. 1979, 51, 100];
2-mono-oxygenase lactate (E.C.1.13.12.4) of Mycobacterium smegmatis which catalyzes the following reaction: EQU L-lactate+O.sub.2 .fwdarw.acetate+CO.sub.2 +H.sub.2 O
combined with a Clark electrode [Mascini et al. Anal. Chem. Acta 1984, 157, 45];
oxidase lactate (E.C.1.1.3.2) of Pediococcus sp., with which the best results are obtained from the point of view of stability, of sensitivity and of reliability and which catalyzes the following reaction:
L-lactate+O.sub.2 .fwdarw.pyruvate+H.sub.2 O.sub.2 combined with detection of the partial oxygen pressure [Mizutani et al. Anal. Chem. 1983, 55, 35] or with detection of H.sub.2 O.sub.2 [Mullen et al. Clin. Chem. Acta 1986, 157, 191].
In order to achieve the objective indicated above, which is that of being able to determine, through direct measurements, the biochemical parameters of the skin, it was interesting to use the known method in accordance with which the sample to be measured was brought into contact with a membrane in which an enzyme is immobilized (as described by Romette, Doctoral Thesis, University of Compiegne, 1986), which enzyme will catalyze the transformation of the substance to be measured with, particularly in the case of L-lactate, oxygen consumption and production of H.sub.2 O.sub.2, with means being provided to detect a phenomenon caused by the consumption of oxygen or the production of H.sub.2 O.sub.2, for example means for amperometric detection of the decrease in partial oxygen pressure (one example being the Clark electrode which is described in more detail below), with the enzymatic membrane being applied to the sensitive end of the electrode.
In effect, the sensitivity of such biosensors is most often considerable. However, the direct application of the electrode on the cutaneous surface has been shown to be impossible, due to difficulties arising from the compression of the electrolyte film, the mechanical resistance of the membrane and the placing in solution of the substrate.